Investigation of tumour protein profiles may allow for identification of cancer markers and potential therapeutic targets. The aim of the present study was to analyse protein profiles derived from fresh-frozen neoplastic and non-neoplastic canine lymph nodes using two-dimensional electrophoresis (2DE), followed by immunohistochemical examination of archival canine lymphoma tissues for identification of differentially expressed protein associated with neoplastic growth. 2DE was used for comparative analysis of proteins extracted from fresh frozen canine B-cell lymphomas (n = 4) and non-neoplastic lymph nodes (n = 6). Liquid chromatography–tandem mass spectrometry was applied for protein identification in selected gel spots. To determine whether elevated expression of protein deglycase DJ-1, which was a protein identified in one of the lymphoma-2DE gel spots, was related to neoplastic cells, archival paraffin wax-embedded tissues of canine lymphomas (n = 20) and non-neoplastic canine lymph nodes (n = 7) were analysed by immunohistochemistry using PARK7/DJ-1 rabbit polyclonal antibody (Abcam).In one of the 2DE gel spots showing more than 4-fold overexpression between the two groups, protein deglycase DJ-1, known as ‘Parkinson disease protein 7’ (PARK7), was identified. Immunohistochemical analysis of PARK7/DJ-1 expression in canine lymphomas revealed diffuse, moderate to strong, nuclear and cytoplasmic immunolabelling in neoplastic cells, but only faint, or absent, immunolabelling in cells of necrotic appearance. Non-neoplastic lymph nodes had weak to moderate cytoplasmic and only occasionally strong nuclear immunolabelling in lymphocytes. The results of proteomic and immunohistochemical analyses allowed for the identification of PARK7/DJ-1 as a protein associated with neoplastic growth in canine lymphoma.